Association of Genetic Polymorphisms with Hepatitis C Virus-Related Liver Cirrhosis in Japan

in vivo 34 : 3309-3313 (2020) doi:10.21873/invivo.12169

Association of Genetic Polymorphisms With Hepatitis C Virus-related Liver Cirrhosis in Japan SHINYA KAMIMURA, AKINORI TAMURA, TOMOTAKA ISHII, TATSUO KANDA and MITSUHIKO MORIYAMA

Division of Gastroenterology and Hepatology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan

Abstract. Background/Aim: Hepatitis C virus (HCV) infection Patients infected with HCV do not always develop is an important health problem in the direct-acting antivirals- cirrhosis (5). It is well known that the frequencies of cirrhosis era. HCV causes life-threatening diseases, such as cirrhosis and HCC are different among different races (6, 7). These and hepatocellular carcinoma. Our aim was to examine facts suggest that certain single-nucleotide polymorphisms whether certain single-nucleotide polymorphisms (SNPs) are (SNPs) affect the development of hepatic fibrosis in patients associated with the prevalence of HCV infections progressing with HCV infection. In the United States, subjects with to cirrhosis in the Japanese population by a genome-wide chronic hepatitis C carrying the DEAD box polypeptide 5 association study-based approach. Materials and Methods: We (DDX5) minor allele or DDX5- DNA polymerase gamma 2, used DNA extracted from blood specimens of Japanese accessory subunit (POLG2) haplotypes are at increased risk subjects with the establishment of the BioBank Japan project. of developing advanced fibrosis, whereas those carrying the Results: We observed statistically significant differences in the carnitine palmitoyltransferase 1A (CPT1A) minor allele are frequency of 4 SNPs (rs1989972, rs2293766, rs1877033 and at decreased risk (8). rs4805439) between anti-HCV-positive cirrhotic patients and It has been reported that compared to a major, wild-type controls. Conclusion: Four SNPs are associated with (WT) CC allele, the minor allele rs4986791 in the TLR4 susceptibility to cirrhosis among HCV-infected Japanese gene encoding the T399I change, confers protection against subjects, while further studies with cohorts other than those fibrosis progression, along with another highly cosegregated sourced from BioBank Japan, must be conducted. SNP (rs4986790) located at codon position 299 (p.D299G) (9), although the TLR4 SNPs (rs4986790 and rs4986791) are Hepatitis C virus (HCV) infection is an important health uniformly distributed in Japanese patients (10). problem in Japan and the United States, although direct- In the present study, we examined whether certain SNPs are acting antivirals (DAAs) against HCV have been introduced associated with the prevalence of HCV infections progressing (1, 2). HCV infection causes acute and chronic hepatitis, to cirrhosis in the Japanese population by a genome-wide cirrhosis and hepatocellular carcinoma (HCC) for hepatic association study (GWAS)-based approach. We used DNA manifestations and cryoglobulinemia, lymphoma, insulin extracted from blood specimens of Japanese subjects with the resistance, type 2 diabetes and chronic kidney diseases for establishment of the BioBank Japan project that stores and extrahepatic manifestations (3, 4). HCC occurs in ~7% of maintains a number of annotated liver disease cases (11). We patients with cirrhosis per year, although HCC occurs in less observed that four SNPs are associated with susceptibility to than 1% of patients with non-advanced liver fibrosis per cirrhosis among HCV infected Japanese subjects. year (3). Materials and Methods

Subjects, DNA preparation and SNP genotyping. Study 1. We selected This article is freely accessible online. patients with cirrhosis (n=195; male/female: 107/88; age, 65±9 years) and control subjects who did not have liver diseases (n=1,553; Correspondence to: Tatsuo Kanda, MD, Ph.D., Associate Professor, male/female: 889/664; age, 61±18 years). The HCV infection status Division of Gastroenterology and Hepatology, Department of of patients with cirrhosis was: 80, positive; 25, negative; and 90, Medicine, Nihon University School of Medicine, 30-1 Oyaguchi- unknown. Control subjects were also enrolled as patients with kamicho, Itabashi-ku, Tokyo 173-8610, Japan. Tel: +81 339728111, diseases other than liver diseases in BioBank Japan (11). Patients who Fax: +81 339568496, e-mail: [email protected] had cirrhosis were also excluded from these controls. A GWAS was performed with high-density oligonucleotide arrays (Perlegen Key Words: BioBank Japan, GWAS, HCV, SNP, TGFBI. Sciences, Santa Clara, CA, USA) for SNP genotyping.

3309 in vivo 34 : 3309-3313 (2020)

Table I . Twenty-nine highly ranked single-nucleotide polymorphisms Table II . Genotyping results of 29 single-nucleotide polymorphisms (SNPs) which were associated with cirrhosis. (SNPs) in patients with cirrhosis.

SNP Chromosome Allele Dominant Recessive SNP Chromosome Allele # [1/1] [1/2] [2/2] Frequency (p-Value) model model [1/2] [1] ## (p-Value) (p-Value) rs17380837 12 C/T 40 36 4 0.725 rs17380837 12 <0.001 <0.001 <0.001 rs4419128 18 A/G 0575 0.031 rs4419128 18 <0.001 <0.001 0.3570 rs2114196 8 C/T 12 39 4 0.394 rs2114196 8 <0.001 <0.001 –1.000 rs3813573 15 G/A 31 38 11 0.625 rs3813573 15 <0.001 <0.001 <0.001 rs3783728 14 T/C 73 610.950 rs3783728 14 <0.001 1.000 <0.001 rs1143637 2 G/A 74 600.963 rs1143637 2 <0.001 0.1672 <0.001 rs1989972 5 C/A 12 33 35 0.362 rs1989972 5 <0.001 <0.001 <0.001 rs1443899 8 T/C 57 21 2 0.844 rs1443899 8 <0.001 <0.001 <0.001 rs2493035 6 T/C 45 27 5 0.760 rs2493035 6 <0.001 <0.001 0.0683 rs4954144 2 T/G 71 810.938 rs4954144 2 <0.001 <0.001 0.0227 rs2116287 10 T/C 4 40 35 0.304 rs2116287 10 <0.001 <0.001 0.0055 rs6555491 5 T/C 73 700.956 rs6555491 5 <0.001 <0.001 0.1647 rs1874361 1 G/T 6 36 36 0.308 rs1874361 1 <0.001 0.0066 <0.001 rs1877033 4 T/C 66 14 0 0.913 rs1877033 4 <0.001 0.1650 <0.001 rs829523 3 C/T 72 610.949 rs829523 3 <0.001 0.0013 0.1153 rs2279674 4 G/T 34 38 6 0.679 rs2279674 4 <0.001 0.0029 0.0069 rs1157977 6 C/A 52 24 3 0.810 rs1157977 6 <0.001 <0.001 0.0081 rs3737964 1 G/A 62 16 1 0.886 rs3737964 1 0.0012 0.0011 0.3832 rs349765 5 A/G 77 300.981 rs349765 5 0.0014 0.0013 –1.000 rs4805439 19 A/G 2 17 53 0.146 rs4805439 19 0.0017 0.0093 0.0106 rs4663603 2 T/C 77 300.981 rs4663603 2 0.0026 0.0050 0.2728 rs10500651 11 T/G 70 10 0 0.933 rs10500651 11 0.0030 0.0114 0.0103 rs2293766 7 C/T 13 28 35 0.459 rs2293766 7 0.0034 0.0016 0.1694 rs674993 3 A/G 36 34 4 0.716 rs674993 3 0.0040 0.0545 0.0105 rs16987220 2 G/A 46 31 3 0.789 rs16987220 2 0.0043 0.0391 0.0126 rs6511152 19 C/G 26 37 16 0.564 rs6511152 19 0.0045 0.0270 0.0168 rs2036100 1 G/C 41 33 6 0.719 rs2036100 1 0.0051 0.0174 0.0249 rs9380236 6 A/G 59 21 0 0.869 rs9380236 6 0.0080 0.0150 0.0364 rs10197150 2 T/C 23 42 9 0.595 rs10197150 2 0.0084 0.1534 0.0087 #Ancestral allele; ## frequency [1]=allele 1/(allele 1+allele 2).

Study 2. We selected patients with cirrhosis (n=753; male/female: implementation of personalized medicine (11). All patients 431/322; age, 63±10.7 years) and control subjects who did not have participating in the present study provided written informed consent liver diseases (n=1,358; male/female: 738/620; age, 59±13.5 years). and the study protocol was approved by the ethics committees of The HCV infection status of patients with cirrhosis was: 95, RIKEN Yokohama Institute and of each participating institution. positive; 110, negative; and 548, unknown. Control subjects were The study protocol for study 3 was also approved by the Ethics enrolled as patients with diseases other than liver diseases in Committee of Nihon University School of Medicine (i-1) and BioBank Japan (11). Patients who had cirrhosis were also excluded conformed to the ethical guidelines of the Declaration of Helsinki. from these controls. A GWAS was performed with a GeneChip SNP array (Affymetrix, Santa Clara, CA, USA) for SNP genotyping. Statistical analysis. Statistical analyses were performed using a Fisher’s exact test. p<0.05 was considered as a statistically Study 3. We performed 29 SNP analyses of anti-HCV-positive significant difference. Statistical analysis was performed with SPSS patients with cirrhosis (n=80; male/female: 45/35; age, 65±11 v.15 (SPSS Inc, Chicago, IL, USA). We analyzed the differences years). DNA samples were genotyped with a TaqMan SNP between the case and control groups in terms of the distribution of genotyping assay (Applied Biosystems Inc, Foster City, CA, USA) genotypes with an allelic model by using a Cox’s proposal hazards using an ABI 7500 Fast real-time PCR system, according to the model analysis. manufacturer’s recommended protocols (10). The PCR was performed as follows: 95˚C for 10 min, followed by 55 cycles of Results 95˚C for 15 sec and 60˚C for 1 min. The subjects did not have HCC at blood sample collection. SNP alleles of Japanese healthy subjects (HapMap-JPT) were used as controls from the International Candidate SNPs associated with cirrhosis selected by the HapMap Project (https://hapmap.ncbi.nlm.nih.gov/index.html) (12). GWAS. A GWAS was performed with high-density BioBank Japan was launched in 2003, establishing a large oligonucleotide arrays (Perlegen Sciences, Santa Clara, CA, Japanese patient-oriented biobank to contribute to the USA), resulting in the selection of 233,820 SNPs for

3310 Kamimura et al : SNPs Associated With HCV-related Cirrhosis

Table III . Frequencies of single-nucleotide polymorphism (SNP) Table V . Frequencies of single-nucleotide polymorphism (SNP) rs1989972 between anti-hepatitis C virus (HCV)-positive subjects with rs1877033 between anti-hepatitis C virus (HCV)-positive subjects with liver cirrhosis (LC) and controls. liver cirrhosis (LC) and controls.

Allele LC Control OR 95% CI p-Value Allele LC Control OR 95% CI p-Value

C/C 33 22 ––0.048 # TC/C 00––0.014 # C/A 35 54 C/T 14 1 A/A 12 10 T/T 66 42 C/C 33 22 2.04 1.06-3.94 0.047 # C/C 00––1 Others 47 64 Others 80 43 A/A 12 10 0.75 0.3-1.84 0.648 T/T 66 42 8.91 1.13-70.3 0.018 # Others 68 76 Others 14 1 Allele C 104 98 1.33 0.86-2.07 0.264 Allele C 14 1 8.15 1.05-63.1 0.022 # Allele A 59 74 Allele T 146 85

OR, Odds ratio; 95%CI, 95% confidence interval; #p<0.05, statistically OR, Odds ratio; 95%CI, 95% confidence interval; #p<0.05, statistically significant difference by Fisher’s exact test. significant difference by Fisher’s exact test.

Table IV . Frequencies of single-nucleotide polymorphism (SNP) Table VI . Frequencies of single-nucleotide polymorphism (SNP) rs2293766 between anti-hepatitis C virus (HCV)-positive subjects with rs4805439 between anti-hepatitis C virus (HCV)-positive subjects with liver cirrhosis (LC) and controls. liver cirrhosis (LC) and controls.

Allele LC Control OR 95% CI p-Value Allele LC Control OR 95% CI p-Value

T/T 33 25 ––0.085 G/G 53 24 ––0.052 T/C 28 43 G/A 17 15 C/C 13 18 A/A 25 T/T 35 25 2.08 1.09-3.98 0.034 # G/G 53 24 2.32 1.05-5.13 0.044 # Others 41 61 Others 19 20 C/C 13 18 1.28 0.58-2.83 1 A/A 70 39 3.59 0.63-20.5 0.103 Others 63 68 Others 24 Allele T 98 93 1.54 0.99-2.41 0.149 Allele G 123 63 2.32 1.21-4.47 0.017 # Allele C 54 79 Allele A 21 25

genotyping, at the first stage. A GWAS was performed with we identified four loci conferring susceptibility to cirrhosis a GeneChip SNP array (Affymetrix, Santa Clara, CA, USA) among HCV-infected subjects. including 233,820 SNPs, resulting in the selection of 2,670 SNPs for genotyping, at the second stage. Finally, we Discussion selected 29 highly ranked SNPs as candidates associated with cirrhosis ( Table I ). In the present study, we used the collection of GWAS data and DNA from HCV-infected cirrhotic individuals and Association of SNPs with HCV infection and cirrhosis in controls from BioBank Japan (11) and used the data of SNP Japanese subjects. Table II shows the frequencies of these alleles of Japanese healthy subjects (HapMap-JPT) as a SNPs in 80 anti-HCV-positive patients with cirrhosis. We control from the International HapMap Project (12). We observed the statistically significant differences in the identified the involvement of four loci in conferring frequencies of 4 SNPs (rs1989972, rs2293766, rs1877033 susceptibility to cirrhosis among HCV-infected subjects. and rs4805439) between anti-HCV-positive cirrhotic patients TGFBI is one of the useful markers to diagnose hepatitis and controls ( Tables II-VI ). Two SNPs (rs1989972 and B virus (HBV)-related cirrhosis (13). Plasma proteome rs2293766) were located within an intron of transforming profiling revealed a strong association between TGFBI, growth factor beta induced (TGFBI) and zonadhesin (ZAN), dipeptidyl peptidase 4 (DPP4), alanyl aminopeptidase, respectively. The other 2 SNPs (rs1877033 and rs4805439) membrane (ANPEP), polymeric immunoglobulin receptor were located outside the protein coding regions. Together, (PIGR) and apolipoprotein E (APOE) and nonalcoholic fatty

3311 in vivo 34 : 3309-3313 (2020) liver disease (NAFLD)-derived cirrhosis (14). TGFBI drives Authors’ Contributions the proliferation of hepatocytes (15). TGFB-induced protein TGFBI is a collagen-associated protein associated with S.K., A.T. and M.M. conceptualized the study., collected data, hepatic fibrogenesis (16). carried out the analysis. S.K., A.T., T.I., T.K. and M.M. drafted the initial manuscript and revised the manuscript. All Authors approved Fibroblast growth factor 19 (FGF-19) treatment increased the final manuscript. the expression of proteins known to drive proliferation [TGFBI, vascular cell adhesion molecule-1 (VCAM-1), Acknowledgements Annexin A2 and high density lipoprotein binding protein (HDLBP)] (15). It is well known that FGF is also a potent Authors thank Prof. Shiro Maeda, Laboratory for Endocrinology, angiogenic molecule involved in HCC progression (17). Metabolism and Kidney Diseases, RIKEN Center for Integrative Recently, Kim et al. reported that FGF-2 and its receptor Medical Sciences, Yokohama 230-0045, Japan and Prof. Naoya SNPs are associated with the survival of patients with HBV- Kato, Laboratory of Molecular Medicine, Human Genome Center, related HCC (18). Although rs1989972 is located within an Institute of Medical Science, University of Tokyo, Tokyo, Japan, for intron of TGFBI, we observed a weak association between valuable discussion. rs1989972 C/C and patients with HCV infection and cirrhosis (Table III). References ZAN is a mosaic-type protein that localizes to the apical 1 Tanaka J, Akita T, Ko K, Miura Y, Satake M and head of spermatozoa (19). The association between ZAN and Epidemiological Research Group on Viral Hepatitis and its hepatic fibrosis is unclear. 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